The goal of this research proposal is to examine mechanisms by which opioid receptor agonists provide protection from ischemia/reperfusion injury when administered at the time of reperfusion and when administered as chronic therapy. We hypothesize that delta and kappa opioid receptor agonists confer cardioprotection when administered prior to reperfusion by activating pro-survival signaling pathways including the PI3K/akt/GSKbeta/ERK 1/2 axis (reperfusion injury salvage kinases or the RISK pathway). With chronic therapy, we hypothesize that sustained cardioprotection is mediated by two complementary mechanisms: 1) up-regulation of cardioprotective proteins including 12-LO, iNOS, and COX-2 (delayed inducible salvage pathway or the DISP pathway), and 2) induction of beta-2 adrenergic receptors resulting in inhibition of adenylyl cyclase. Overall, we predict that the sarcolemmal isoform of the ATP-sensitive potassium (KATP) channel is the trigger and the mitochondrial KATP channel is the end-effector of opioid receptor-induced cardioprotection. Our studies involve the use of an in vivo rat/mouse model of infarction and an isolated mouse heart model of global ischemia and reperfusion in which infarct size and recovery of left ventricular contractile function are used as indices of ischemic injury, respectively. The involvement of specific signaling pathways, cardioprotective proteins, and receptor subtypes will be assessed by using selective pharmacological probes, gene "knock-out" mice, Western immunoblotting, radioligand binding analysis, and recording of sarcolemmal (patch clamp recordings of isolated cardiac myocytes) and mitochondrial (recordings of KATP channel currents in mitochondrial lipid bi-layers) KATP channel currents. Collectively, these studies combine state-of-the-art techniques to provide new information regarding mechanisms by which opioid receptor agonists provide protection from ischemia/reperfusion injury. Information gained from these studies will hopefully lead to new therapies to treat patients with ischemic heart disease.